Ethanol production by a mixed culture of flocculent strains of Zymomonas mobilis and Saccharomyces sp.

 Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l^-1 h^-1. No levan was produced even if a sucrose-based medium was used. Received: 20 April 1995/Received revision: 26 July 1995/Accepted: 13 September 1995     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1: 1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola. Received: 29 July 1998 / Received revision: 5 October 1998 / Accepted: 11 October 1998     

Pikas (Ochotona princeps : Lagomorpha) as allogenic engineers in an alpine ecosystem

Pikas (Ochotona princeps: Lagomorpha) build caches of vegetation (“haypiles”), which serve as a food source during winter in alpine and subalpine habitats. Haypiles appear to degrade over time and form patches of nutrient-rich soils in barren talus and scree areas. We sampled soils underneath and next to haypiles, and plants growing on and near haypiles in an alpine cirque in northwestern Wyoming, USA, to determine the effects of pika food caches on N, C, and C/N ratios in soils and plants. We found that (1) haypile soils had significantly higher carbon and nitrogen levels and lower C/N ratios than both adjacent soils and soils in the general study area, (2) two of three plant species tested (Polemonium viscosum and Oxyria digyna) had significantly higher levels of tissue percent N when growing on haypile soils, and (3) total standing plant biomass at the study site increased with soil percent N suggesting that vegetation was nitrogen limited. Pikas may therefore function as allogenic ecosystem engineers by modulating nutrient availability to plants. Received: 5 July 1997 / Accepted: 30 November 1997     

Lipid storage compounds in marine bacteria

Forty psychrophile or psychrotrophic crude-oil-utilizing marine bacteria were investigated for their ability to accumulate lipid storage compounds in the cytoplasm during cultivation under nitrogen-limiting conditions. Most of them (73%) were able to accumulate specialized lipids like polyhydroxyalkanoic acids (PHA) while other lipids such as wax esters occurred in two isolates. Accumulation of PHA occurred predominantly at low temperatures (4–20 °C) as demonstrated for three isolates. Electron microscopy revealed polyphosphate inclusions occurring in two isolates in addition to PHA. Cells of the isolate Acinetobacter sp. 211 were able to synthesize and accumulate lipid inclusions during growth on acetate, ethanol, olive oil, hexadecanol and heptadecane. The composition of the lipid inclusions depended on the compounds provided as carbon source. Wax esters and acylglycerols occurred mainly during the cultivation on olive oil; in contrast, wax esters and free alcohols occurred during cultivation on hexadecanol. Total fatty acids in cells of the Acinetobacter sp. 211 amounted to 25% of the cellular dry weight in olive-oil-grown cells. Palmitic acid was the main fatty acid in the lipids when the cells were cultivated on acetate or ethanol (44% and 32% of total fatty acids respectively). In contrast, fatty acids occurring in the lipids during cultivation on hexadecanol, heptadecane or olive oil were related to the carbon source. The fatty acids present in the accumulated lipids consisted predominantly of saturated and unsaturated straight-chain fatty acids with a chain length ranging from 12 to 18 carbon atoms. Analysis of the lipid-granule-associated proteins in cells of Acinetobacter sp. 211 revealed a protein of 39 kDa as the predominant protein species. Received: 2 July 1996 / Received revision: 3 September 1996 / Accepted: 28 September 1996     

Degradation of phosphonates by streptomycete isolates

Wild-type Streptomyces sp. strains, able to utilise both naturally occurring and synthetic organophosphonates, were isolated. High levels of inorganic phosphate were necessary for their growth in complete medium as well as in medium, supplemented with phosphonates as the sole carbon or nitrogen source. Isolate StA expressed detectable enzymatic activity against 2-aminoethylphosphonate in vivo. Streptomycete StC had a surprising ability to degrade N-phosphonomethylglycine (glyphosate) in a phosphate-independent manner via C–P bond cleavage accompanied by sarcosine formation. Received: 5 January 1999 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Identification of Rhodococcuserythropolis isolates capable of degrading the fungicide carbendazim

Continuous enrichment cultures were used to identify bacterial isolates capable of degrading the fungicide carbendazim. The bacteria originated from sites that had been repeatedly treated with the structurally related parent fungicide, benomyl, over a period of several years. Six isolates were identified as carbendazim degraders on the basis of their ability to produce diffusion-clearing zones on a minimal salts solid medium spray-coated with a 0.1% solution of carbendazim, their ability to grow in a minimal salts broth supplemented with carbendazim as the sole carbon source, and their ability to reduce carbendazim levels in liquid cultures. All six isolates were identified as Rhodococcus erythropolis or a closely related species by analyses of nutritional utilization and whole-cell fatty acid methyl ester profiles. A chemically induced mutant of R. erythropolis isolate B2E was identified that was no longer capable of degrading carbendazim, as determined by negative results in all three assays. Further characterization of these strains provides an opportunity for their development in bioremediation of the compound. Received: 22 July 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene to degradation by pure cultures of 1,1-diphenylethylene-degrading aerobic bacteria

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) is the peri-chlorinated derivative of 1,1-diphenylethylene (DPE). Biodegradation of DDE and DPE by bacteria has so far not been shown. Pure cultures of aerobic bacteria involved in biodegradation of styrene and polychlorinated biphenyls (PCB) were therefore screened for their ability to degrade or cometabolize DPE and DDE. Styrene-metabolizing bacteria (Rhodococcus strains S5 and VLB150) grew with DPE as their sole source of carbon and energy. Polychlorinated-biphenyl-degrading bacteria (Pseudomonas fluorescens and Rhodococcus globerulus) were unable to degrade DPE even in the presence of an easily utilizable cosubstrate, biphenyl. This is the first report of the utilization of DPE as sole carbon and energy source by bacteria. All the tested bacteria failed to degrade DDE when it was provided as the sole carbon source or in the presence of the respective degradable cosubstrates. DPE transformation could also be detected in cell-free extracts of Rhodococcus S5 and VLB150, but DDE was not transformed, indicating that cell wall and membrane diffusion barriers were not limiting biodegradation. The results of the present study show that, at least for some bacteria, the chlorination of DDE is the main reason for its resistance to biodegradation by styrene and DPE-degrading bacteria. Received: 28 May 1997 / Received revision: 28 October 1997 / Accepted: 31 October 1997     

Degradation and bioconversion of aliphatic and aromatic hydrocarbons by Rhodococcus ruber 219

 A tetrahydrofuran-degrading bacterial strain, which had previously been tentatively assigned as Rhodococcus sp. strain 219, has now been identified as Rhodococcus ruber using physiological and chemotaxonomical tests. A comparison with the type strain DSM 43338 has revealed that the new strain differs in its ability to degrade or convert tetrahydrofuran and compounds of similar structure such as 2,5-dimethyltetrahydrofuran or tetrahydropyran. Tetrahydrofuran acts as an inducer for its degradation. When tetrahydrofuran-induced cells were incubated with 2,5-dimethyltetrahydrofuran two primary metabolites could be detected by gas chromatography, and 2-hydroxyhexane-5-one and hexane-2,5-dione were isolated and characterized by ¹H-NMR spectroscopy or as dinitrophenylhydrazones. The formation of these intermediates is consistent with an initial 2hydroxylation of the cyclic ether, which has not yet been described in microorganisms. Received: 19 July 1995/Received last revision: 31 October 1995/Accepted: 6 November 1995     

Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation,production and characterization

 Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions, is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers. The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa molecular mass range. Received: 7 June 1995/Received revision: 14 September 1995/Accepted: 20 September 1995     

Thermococcus peptonophilus sp. nov., a fast-growing,extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents

Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7–2 μm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85–90°C (range 60–100°C), pH 6 (range pH 4–8), a NaCl concentration of 3% (range 1–5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005–5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Received: 8 March 1995 / Accepted: 24 May 1995     

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 °C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons. Received: 27 December 1996 / Received last revision: 10 June 1997 / Accepted: 14 June 1997     

Identification and production of 3-hydroxy-Δ9-cis-1,18-octadecenedioic acid by mutants of Candida tropicalis

 The transformation of oleic acid by mutants of Candida tropicalis was studied in fed-batch cultures. Besides Δ⁹-cis–1,18-octadecenedioic acid, 3-hydroxy-Δ⁹-cis–1,18-octadecenedioic acid was detected as the main fermentation product. Here we describe the production, isolation and the complete chemical characterization of the purified 3-hydroxy-Δ⁹-cis–1,18-octadecenedioic acid. The geometric configuration of the double bond was not changed during bioconversion. The enantiomeric excess of the compound was 76%. Mutagenesis of C. tropicalis DSM 3152 with N-methyl-N-nitro-N′-nitrosoguanidine and selection with oleic acid as the sole carbon source led to mutant M 25, which produced the 3-hydroxy-Δ⁹-cis–1,18-octadecenedioic acid at a 1.8-fold higher concentration in the medium as compared to the parent strain. The maximum concentration of the hydroxy dioic acid was 19.4 g/l after 223 h fermentation. Received: 24 August 1995/Received revision: 21 September 1995/Accepted: 4 October 1995     

Biodegradation of methyl t-butyl ether by pure bacterial cultures

Three pure bacterial cultures degrading methyl t-butyl ether (MTBE) were isolated from activated sludge and fruit of the Gingko tree. They have been classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter. These cultures degraded 60 ppm MTBE in 1–2 weeks of incubation at 23–25 °C. The growth of the isolates on MTBE as sole carbon source is very slow compared with growth on nutrient-rich medium. Uniformly-labeled [¹⁴C]MTBE was used to determine ¹⁴CO₂ evolution. Within 7 days of incubation, about 8% of the initial radioactivity was evolved as ¹⁴CO₂. These strains also grow on t-butanol, butyl formate, isopropanol, acetone and pyruvate as carbon sources. The presence of these compounds in combination with MTBE decreased the degradation of MTBE. The cultures pregrown on pyruvate resulted in a reduction in ¹⁴CO₂ evolution from [¹⁴C]MTBE. The availability of pure cultures will allow the determination of the pathway intermediates and the rate-limiting steps in the degradation of MTBE. Received: 8 December 1995 / Received last revision: 5 August 1996 / Accepted: 12 August 1996     

Cell-free extract(s) of Pseudomonas putida catalyzes the conversion of cyanides,cyanates, thiocyanates,formamide, and cyanide-containing mine waters into ammonia

 Our isolate, Pseudomonas putida, is known to be capable of utilizing cyanides as the sole source of carbon (C) and nitrogen (N) both in the form of free cells and cells immobilized in calcium alginate. In the present study, the cell-free extract(s) were prepared from the cells of P. putida grown in the presence of sodium cyanide. The ability of enzyme(s) to convert cyanides, cyanates, thiocyanates, formamide and cyanide-containing mine waters into ammonia (NH₃) was studied at pH 7.5 and pH 9.5. The kinetic analysis of cyanide and formamide conversion into NH₃ at pH 7.5 and pH 9.5 by the cell-free extract(s) of P. putida was also studied. The K _m and V _max values for cyanide/formamide were found to be 4.3/8 mM and 142/227 μmol NH₃ released mg protein^-1 min^-1 respectively at pH 7.5 and 5/16.67 mM and 181/434 μmol NH₃ released mg protein^-1 h^-1 respectively at pH 9.5. The study thus concludes that the cell-free extract(s) of P. putida is able to metabolize not only cyanides, cyanates, thiocyanates, and formamide but also cyanide-containing mine waters to NH₃. Received: 10 April 1995/Received revision: 24 July 1995/Accepted: 22 August 1995     

Growth and production of xylanolytic enzymes by the extreme thermophilic anaerobic bacterium Thermotoga thermarum

 Cultivation of the extreme thermophilic anaerobic bacterium Thermotoga thermarum at 77°C on xylan was accompanied by the formation of heat-stable endoxylanase (136U/l), β-xylosidase (44U/l) and α-arabinofuranosidase (10U/l). These enzymes were mainly associated with the cells and could not be released by detergent treatment {0.1–1.0mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS)}. Endoxylanases with a molecular weight of 40, 83 and 100kDa were induced when xylan or xylose were used as substrates for growth. In the presence of other sugars like glucose, maltose, arabinose or starch, low concentrations of the low-molecular-weight endoxylanase (40kDa) was detected. Xylose was found to be the best substrate for the induction of β-xylosidase and α-arabinofuranosidase but not for growth. Cultivation of T. thermarum in a dialysis batch fermentor resulted in a significant increase in cell concentration and enzyme level. A total cell count of 1.3×10⁹ cells/ml and 202U/l of endoxylanase were measured when partially soluble birchwood xylan was used as the carbon source. The use of insoluble beechwood xylan as the substrate caused the elevation of the maximal cell concentration and enzyme level up to 2.0×10⁹ cells/ml and 540U/l, respectively. Received: 14 September 1995/Received revision: 15 December 1995/Accepted: 18 December 1995     

Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol

 Pseudomonas sp. strain M285 immobilized on diatomaceous earth beads was used to remove 3,5,6-trichloro-2-pyridinol (TCP) from industrial wastewater. Batch studies showed that immobilized Pseudomonas sp. strain M285 mineralized [2,6-¹⁴C]TCP rapidly; about 75% of the initial radioactivity was recovered as ¹⁴CO₂. Transformation of TCP was inhibited by high concentrations of salt, and addition of osmoprotectants (proline and betaine at 1 mM) did not reduce the adverse effect of salt. TCP-containing wastewater (60–140 mg/l) was passed through columns containing immobilized Pseudomonas sp. strain M285 at increasing flow rates and increasing TCP concentrations; TCP removal of 80%–100% was achieved. Addition of nutrients, such as glucose and yeast extract, retarded TCP degradation. Growing cell cultures were found to be better inocula for immobilization than resting cells. Received: 5 February 1996 / Received last revision: 12 August 1996 / Accepted: 24 August 1996     

The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant

 The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N- and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N- and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-β-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U⋅mg^-1, 5.5 and 102^°C, respectively. The endoxylanase was stable at 90^°C and retained 50% activity when incubated for 2 h at 100^°C. Received: 19 May 1995/Received revision: 31 July 1995/Accepted: 7 September 1995     

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis

 A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10hydroxystearic acid. Received: 17 July 1995/Received last revision: 24 October 1995/Accepted: 30 October 1995     

Enzymatic Dehalogenation of Pentachlorophenol by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> of the Microbial Community from Tannery Effluent

Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in the chemostat. The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-ρ-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC). Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production. PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography. The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24,000 Da. Received: 22 July 2002 / Accepted: 23 September 2002